A method for achieving regeneration of soybean and its relatives from somatic tissue cultures has long been sought. Unlike such easily regenerable species as tobacco and petunia, soybean has resisted prior attempts to regenerate whole plants from somatic tissues. Such methods are very desirable in allowing the induction of desirable traits into soybean or species capable of breeding therewith (such as G. soja) via somaclonal variation. Such methods would also be of benefit to genetic engineers in allowing transformation of cells by infection with Agrobacteria or by other means resulting in transformed cells in culture containing foreign (heterologous) DNA which could then be regenerated into whole plants bearing seed and expressing foreign genes.
Few methods for regenerating Glycine subgenus soja (comprising G. max (soybean) and G. soja) have been developed, although greater success has been achieved with wild relatives such as G. canescens and G. clandestina. See P. A. Lazzeri, et al. (1985) "A Procedure for Plant Regeneration from Immature Cotyledon Tissue of Soybean," Plant Molecular Biology Reporter Vol. 3, No. 4, 160-167, and D. F. Hildebrand, et al. (1986) "Soybean [Glycine max (L.) Merr.]," Biotechnology in Agriculture and Forestry Vol. 2: Crops I (Y. P. S. Bajaj, ed.) 283-308.
Most manipulations of Glycine species described in the literature involving embryogenesis provide a basal medium containing an auxin as an induction medium for the development of embryoids. After embryo formation, the embryos may be moved to a maturation medium and then to a medium containing cytokinin and reduced auxin for shooting. However the desirability of using NAA in high concentrations or synergistically lowered concentrations of carbohydrates and auxins has not previously been recognized as an aid in attaining high frequencies of normal somatic embryos. Further, specific cotyledonary cells giving rise to somatic embryos in media containing any auxin have not been identified and isolated so as to provide high efficiency in a regeneration method of G. max or other Glycine species.
W. D. Beversdorf, et al., in "Degrees of Differentiation Obtained in Tissue Cultures of Glycine Species," (1977) Crop Sci. Vol. 17, 307-311, first reported somatic embryogenesis, but none of the embryos "germinated." Using an induction medium containing 2,4-D (2,4-dichlorophenoxyacetic acid) and/or NAA (alpha-naphthaleneacetic acid) and 2% sucrose to culture hypocotyl or mature cotyledon tissues or apices, or embryos, of G. max and several related species, Beversdorf, et al. achieved embryo-like structures on some cultivars, but no further development into plantlets. Cotyledon tissues from developing embryos of 56 soybean cultivars developed calli and "non-callus" structures on a medium containing 2 mg/l 2,4-D and 2 mg/l NAA. None of these developed further however.
T. Y. Cheng, et al. (1980), "Plant Regeneration from Soybean Cotyledonary Node Segments in Culture," Plant Sci. Lett. Vol. 19, 91-99, report the stimulation of multiple shoot-bud formation of soybeans in culture using conditioned cotyledonary node segments. The medium used contained 3% sucrose and 0.25 .mu.M of the auxin IBA (indole butyric acid). This method did not involve the use of somatic tissues, but rather used explants consisting of totipotent cells to evaluate the effectiveness of various media. It is not clearly reported that whole plants capable of independent growth in soil were regenerated.
H. Saka, et al. (1980), "Stimulation of Multiple Shoot Formation on Soybean Stem Nodes in Culture," Plant Sci. Lett Vol. 19, 193-201, similarly describe the formation of shoot-buds on stem nodes or apices of G. max using a culture medium containing the auxin IBA and 3% sucrose or other carbohydrate. This work did not involve the use of somatic tissues, but rather used tissues normally competent to produce shoots to evaluate the effectiveness of various media for producing growth. Whole plant regeneration was not reported.
T. Kameya, et al. (1981), "Plant Regeneration from Hypocotyl Sections of GlYcine Species," Plant Sci. Lett., Vol. 21 289-294, disclose the use of hypocotyl sections of G. canescens and G. tomentella cultured on MS medium supplemented with NAA and BA (6-benzylamino purine) at various concentrations to regenerate normal plants. From the eight species tested including G. max and G. soja regeneration of shoots at high frequency was observed only from hypocotyl sections of G. canescens using 1-5 mg/l BA and 0.1 to 2 mg/l NAA. Whole plants were regenerated.
G. C. Phillips, et al. (1981), "Induction and development of somatic embryos from cell suspension cultures of soybean," Plant Cell Tissue Organ Culture, Vol. 1, 123-129 reported obtaining a single shoot from an embryogenic G. soja suspension culture. Various auxins were evaluated in combination with basal L2 and SL2 media, including NAA at 0.1 to 13.4 .mu.M (approximately 0.02 to 2.7 mg/l). Sucrose concentrations from 2.5% to 12.5% were used. The single shoot obtained came from a G. soja culture grown on an SL2 medium containing 2.25 .mu.M 2,4-D (0.45 mg/l), and transferred to an L2 medium containing the same amount of 2,4-D, plus cytokinin, antiauxin and gibberellin biosynthesis inhibitor.
K. K. Kartha, et al. (1981) "Plant Regeneration from meristems of grain legumes: soybean, cowpea, peanut, chickpea and bean," Can. J. Bot. Vol. 59, 1671-1679, describe plant regeneration from shoot apical meristems of soybean on a medium containing 3% sucrose and 1 .mu.M/l NAA. Whole plants were regenerated; however, this article does not disclose the use of somatic tissue to give rise to new plants, but rather the normal continued differentiation of meristem tissue. The reference is primarily concerned with the determination of optimal growth media for this purpose.
B. D. Reynolds, et al. in an abstract for a presentation at the 79th Annual Meeting of the American Society for Horticultural Science in Ames, Iowa August 8-13, 1982, entitled "Production of Embryoids from Primary and Callus Explants of Soybean," reported production of embryoids from primary explants (hypocotyl, leaf, and root) of Glycine max when cultured on various media. This abstract does not define the media clearly nor provide enabling details. No claim to plant regeneration beyond the embryoid stage is made.
M. L. Christianson, et al. (1983), "A Morphogenetically Competent Soybean Suspension Culture Vol. 222, 632-634, report the regeneration of plantlets from pieces of the embryonic axis of G. max using as an embryo induction medium MS (Murashige and Skoog) medium with the nitrogen salts therein replaced by 20 .mu.M ammonium citrate and also containing 5 mg/l 2,4-D or IAA (indole-3-acetic acid). This reference discloses that the nitrogen substitution was critical to the method. Only one exceptional piece of tissue formed embryos. This, therefore, may have been an accidental and non-reproducible event, and in fact the literature does not report this work having been successfully repeated. Transfer of the embryoids to a medium containing 0.005 mg/l IBA and 0.2 mg/l BA gave rise to shoot formation. Transfer of the shoots to a basal medium containing 0.1 mg/l IAA resulted in root formation to produce plantlets.
U.S. Pat. No. 4,548,901 to Christianson, et al. is based on the above described work and claims an improvement in a method for producing a morphogenetically competent plantlet regeneration culture wherein explants of a large-seeded legume plant are successively cultured and selectively transferred, comprising culturing in a medium containing exogenous auxin and ammonium salt, said medium being free of nitrate ion. This patent also claims a method for generating legume bipolar embryoids which comprises several culturing steps including the use of the medium containing exogenous auxin and an ammonium salt, said medium being free of nitrate ion. The examples of this application are as described in the above article except that the mention of rooting is couched in the present tense, apparently indicating that rooting had not been achieved at the time of filing the patent application.
Neither the Christianson, et al. article nor the Christianson, et al. patent disclose or claim the regeneration of whole plants capable of independent growth in soil, nor capable of seed production.
J. M. Widholm, et al. (1983), "Shoot Regeneration from Glycine canescens Tissue Cultures," Plant Cell Reports, Vol. 2, 19-20, report shoot induction from calli obtained from cotyledons and hypocotyls of G. canescens using several media including media containing 0.5 mg/l NAA and 3% sucrose. Whole plants were not regenerated, and root formation was infrequent.
O. L. Gamborg, et al. (1983), "Somatic Embryogenesis in Cell Cultures of Glycine Species," Plant Cell Reports, Vol. 2, 209-212, report somatic embryogenesis from cell suspension culture of hypocotyl tissue in several Glycine species including three cultivars (out of seven tested) of Glycine max. The embryoid induction medium consisted of the major salts of SL, the micronutrients and vitamins of B5, 10 mg/l casamino acids, 15 .mu.M adenine sulfate, 0.2 .mu.M Picloram (0.04 mg/l) and 0.025-00.25 .mu.M AMO 1618. It was discovered that picloram was most effective for embryoid induction but that it could be replaced by 0.5 to 2.0 .mu.M 2,4-D (0.1 to 0.4 mg/l). No embryoids were induced when NAA was used as the auxin. After embryoids were induced they were transferred to an SL growth medium containing cytokinins. Roots, but no shoots were formed.
European Patent Application No. 85109344.3 by Sungene Technologies Corp., published Feb. 19, 1986, discloses a process for regenerating soybeans comprising the use of four separate media, an embryoid inducing medium, an embryoid maturation medium, a shooting medium, and a rooting medium. The embryo induction medium is disclosed as containing 0.5 to 10 mg/l 2,4-D or 1.0 to 3.0 mg/l IAA plus 3.0 to 10 mg/l 2,4-D and 2% to 3% sucrose. The examples do not definitely disclose that root formation was attained; the claims are directed to "plantlets."
B. J. Li, et al. (1985), "Somatic embryogenesis and plantlet regeneration in the soybean Glycine max", Plant
Cell Reports, Vol. 4, 344-347, report that they obtained plantlets from single cells taken from immature soybean embryos. It is disclosed that it is necessary to first freeze the pods in liquid nitrogen then transfer them to a 60 degree C. water bath for 20 minutes. The immature embryos are dissected and cut into small segments for culturing. The filtering techniques used to obtain single cells from the cultures thus produced were reported as 95% effective. The embryo induction medium contained 2% sucrose and 1 to 2 mg/l 2,4-D. The regeneration of plantlets was described, but no regeneration of whole plants was reported. Applicants herein have attempted to repeat this work without success, as the freezing and rewarming of tissue kills it or destroys its ability to grow further.
B. Lippmann, et al. (1984), "Induction of somatic embryos in cotyledonary tissue of soybean, Glycine max L. Merr.," Plant Cell Reports, Vol. 3, 215-218, describe the use of immature cotyledons from G. max to form embryos in a medium containing 0.5 to 1 mg/l 2,4-D and .25 to 2% sucrose. A few cases of shoot differentiation and root formation were observed with L2 medium containing 0.5 .mu.M/l zeatin. No embryo formation was observed when NAA was used as the auxin rather than 2,4-D. No embryos were formed on media containing sucrose above 2% or glucose above 1.5%. No regeneration of whole plants was reported.
J. E. Grant (1984), "Plant regeneration from cotyledonary tissue of Glycine canescens, a perennial wild relative of soybean," Plant Cell Tissue Organ Culture, Vol. 3, 169-173, reports that cotyledon tissue from immature embryos of G. canescens was induced to form embryos using MS media containing 0.1 .mu.M NAA (0.02 mg/l) and 3% sucrose. This work purports to describe the first whole plant regeneration from a Glycine species, but does not disclose regeneration of G. max.
J. P. Ranch, et al. (1985), "Plant Regeneration from Embryo-Derived Tissue Cultures of Soybeans, In Vitro Cellular & Developmental Biology, Vol. 21, No. 11, 653-658 describe the use of immature G. max and G. soja embryos and cotyledons dissected therefrom to produce embryos which were regenerated into whole fertile plants. The embryo induction medium used was MS medium containing 22.5 to 45.2 .mu.M 2,4-D (5.0 to 10.0 mg/l) and 3% sucrose. An embryo maturation medium, B5 plus IBA and ABA, was used prior to transfer to a germination medium. Whole fertile plants were developed. No use of NAA as an induction medium was reported. Applicants do not concede that this publication may be properly applied as prior art against their invention.
C. A. Newell, et al. (1985) "Protoplast culture and plant regeneration in Glycine canescens, Plant Cell Tissue Organ Culture, Vol. 4, 145-149 describe the regeneration of whole plants of G. canescens from protoplasts taken from hypocotyl tissue. The embryogenesis induction medium contained BA at 0.4 mg/l and NAA at 0.1 and 1.0 mg/l in some experiments reported. The basic medium consisted of R-Medium major salts and CL-Medium osmoticum, and contained 6.84 mg/l sucrose plus 25 mM each of mannitol, sorbitol, xylitol and inositol.
U. B. Barwale, et al. (1986), "Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis," Planta, Vol. 167, 473-481, disclose the use of immature soybean embryos to obtain embryos on an MS medium containing 43.0 .mu.M NAA (8.9 mg/l) and 3% sucrose. Intact embryos were used including both cotyledons and embryonic axes. Whole plants were regenerated.
Master's Thesis by Usha B. Barwale, "Screening of Soybean Cultivars for Plant Regeneration Potential & Regeneration of Soybean Plants from Undifferentiated Tissue," catalogued by the University of Illinois Library Mar. 16, 1986, at page 59 and following, discloses the culturing of whole immature embryos to produce embryogenic calli. The media described contained 3% or more carbohydrate and up to 12 mg/l NAA.
H. R. Kerns, et al. (1986), "Correlation of cotyledonary node shoot proliferation and somatic embryoid development in suspension cultures of soybean (Glycine max L. Merr.), "Plant Cell Reports, Vol. 5, 140-143 disclose the induction of embryos on tissue derived from hypocotyl and cotyledon tissues from germinated seeds using a suspension medium containing 6% sucrose and 0.4 mg/l 2,4-D. No regeneration of the embryos into whole plants was reported.
Regeneration of transformed Glycine species plants has not been previously reported, however, several articles discuss Agrobacterium-Glycine interactions.
H. C. Pedersen, et al. (1983), "Induction and in vitro culture of Soybean Crown Gall Tumors," Plant Cell Reports, Vol. 2, 201-204, discuss the first successful infection of G. max plants with Agrobacteria by enclosing the inoculation site to prevent dehydration. Except for some sporadic emergence of roots from the transformed callus tissue, this article states the transformed tissue did not develop morphological structures, and attempts to induce regeneration with BAP and NAA were unsuccessful.
E. E. Hood et al. (1984) "Restriction Endonuclease Map of pTiBo542, a Potential Ti Plasmid Vector for Genetic Engineering of Plants", Bio/Technology 2:702-709, describe Agrobacterium infection of soybean (cv. Wayne) utilizing strain A281. A later article by E. E. Hood et al. (1986) "T-DNA and Opine Synthetic Loci in Tumors Incited by Agrobacterium tumefaciens A281 on Soybean and Alfalfa Plants, J. Bacteriol. 168:1283-1290 discloses that T-DNA in Agrobacterium infected soybean is of a different length than that in alfalfa.
W. Lranzheng, et al. (1984), "Tumor Induction and Gene Transfer in Annual Species of Glycine by Agrobacterium tumefaciens, Proceedings of the World Soybean Research Conference II, Ames, Iowa, 1984, 195-198 describe attempts to induce tumors by infection of 984 varieties of G. max as well as large numbers of varieties of other Glycine species by inoculation with cultures of fifteen strains of Agrobacterium tumefaciens. Out of 3137 plants of G. max treated, tumors were induced on only four. No attempts at plant regeneration were made.
R. Wyndale et al. (1985), "Dynamics of Endogenous IAA and Cytokinins During Growth Cycle of Soybean Crown Gall and Untransformed Callus," Plant Cell Physiol. 26:1145-1154, described Agrobacterium infection and tumor formation in soybean and found high levels of cytokinin in gall tissues.
L. D. Owens et al. (1985), "Genotypic Variability of Soybean Response to Agrobacterium Strains Harboring the Ti or Ri Plasmids," Plant Physiol. 77:87-94, describe response of various soybean genotypes to Agrobacterium infection via tumor formation and opine synthesis.
In a non-enabling report by H. Bialy (1985), "Soybean Transformed; New Role for cGMP", Bio/Technology 3:200-201, a description is given of expression in soybean cell culture of a kanamycin resistance gene transferred via a Ti plasmid. No regeneration was reported.
D. T. Kudirka, et al. (1986), "Interactions of Agrobacterium tumefaciens with leaf explants in tissue culture," Can. J. Genet. Cytol. 28:808-817, discloses soybean wound tissues remain susceptible to Agrobacterium infection for only four hours after wounding.
R. B. Simpson et al. (1986), "A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes," Plant Molecular Biology 6:403-415, discloses the use of the vir region from A. rhizogenes in A. tumefaciens in a binary vector system to transform soybean (with low efficiency of producing transformed hairy roots). E. A. Shahim and R. B. Simpson, Abstract for presentation at 1986 Conference on Molecular & Cellular Biology of the Soybean, held at Ames, Iowa, entitled "Introduction and Expression of Foreign Genes Into Soybean Via Agrobacterium Rhizogenes" summarizes the work disclosed in the article and states that somatic embryos were formed from transformed soybean root. This abstract also speculates that rhizogenes induced hairy roots can regenerate into whole plants but does not indicate that whole plant regeneration had been achieved or enable such regeneration.
L. D. Owens, et al. (1985) "Genotypic Variability of Soybean Response to Agrobacterium Strains Harboring the Ti or Ri Plasmids," Plant Physiol. Vol. 77, 87-94, describe the effect of G. max and G. soja genotype and maturity on susceptibility to infection by Agrobacterium. No regeneration of plants from infected tissue is described. Infection was achieved by spreading 10 ml of a bacterial suspension containing 5.times.1010 cells/ml on wounds between nodes two and three of plants aged two to three weeks old.
D. Facciotti, et al. (1985), "Light-inducible Expression of a Chimeric Gene in Soybean Tissue Transformed with Agrobacterium," Biotechnology, Vol. 3, 241-246, describes transformation of young soybean plants by injection with Agrobacterium tumefaciens containing a kanamycin resistance gene linked to the 5' portion of a soybean small subunit carboxylase gene. Expression of the kanamycin resistance gene in transformed tumorous callus tissue was obtained. No regeneration of transformed tissue was reported.
M. C. Byrne et al. (1987), "Strain and Cultivar Specificity in the Agrobacterium-Soybean Interaction," Plant Cell, Tissue and Organ Cultures 8:3-15, discusses the responses of various soybean genotype to various Agrobacterium strains in terms of tumor formation and describes the expression of kanamycin resistance in transformed tumor tissue.
None of the foregoing art describes somatic embryogenesis and regeneration of whole soybean plants using NAA, or of any Glycine species using auxins at concentrations as high as 15 mg/l or synergistic combinations of carbohydrates at less than 2% and auxins at low percents. Further, none of the art discloses regeneration of a whole transformed plant. Additionally there is no suggestion or disclosure in the art that regeneration efficiencies high enough for effective transformation can be achieved through contacting selected, particularly embryogenic, cells with the DNA to be transferred, nor that such transformation might be accomplished without the use of a selectable marker such as an antibiotic resistance gene.